Metastable intermediates in myoglobin at low pH.
Resonance Raman and optical absorption spectra of ligand-free (deoxy) myoglobin and CO-bound myoglobin (MbCO) at pH 2.6 have been measured by using continuous-flow/rapid-mixing techniques. The spectra of deoxy myoglobin at low pH within 6 ms of the pH drop demonstrate that the iron-histidine bond has been ruptured but that the heme is still five-coordinate. Comparison with data from model complexes indicates that a weak-field ligand, such as a water molecule, is coordinated at the fifth position. The Raman spectrum of MbCO at low pH has an Fe-CO stretching mode that is characteristic of a six-coordinate heme with an unhindered Fe-CO moiety. Immediately following the pH drop in this case, there is no indication that the iron-proximal histidine bond is broken. Three different structural changes are detected at low pH: (i) the iron-proximal histidine (F8) bond in ligand-free myoglobin is broken and replaced by a weak-field ligand, (ii) the distal pocket in MbCO is opened, and (iii) protein constraints on the heme group in MbCO are relaxed. Previous conclusions that the kinetics of CO-binding in hemoproteins at low pH is modified by rupturing the iron-proximal histidine bond are supported by these new results which, however, demand a more complete reevaluation of the phenomenon.
Han
S
S
AT&T Bell Laboratories, Murray Hill, NJ 07974.
Rousseau
D L
DL
Giacometti
G
G
Brunori
M
M
D000818:Animals; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009211:Myoglobin / Q000378:metabolism*; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods; D014907:Whales
D008024:Ligands; D009211:Myoglobin; C018013:carboxymyoglobin